Introduction

The rise in consumer demand for convenient, ready-to-eat, and health-conscious food options has led to an increase in the consumption of fresh foods like salads, seafood, meat, and horticultural products.¹ Salads have gained substantial popularity among health-conscious individuals in contemporary times. These wholesome food preparations are deemed essential, accompanied by salad dressings, which play a pivotal role. Typically, salad dressings consist of a combination of eggs and oil, along with the potential inclusion of additional components such as vinegar, sugar, salt, mustard, pepper, or various spices.² Eggs, renowned for their exceptional nutritional value and economical nature, stand as one of nature’s most formidable offerings. However, it is crucial to exercise caution when consuming salad cream derived from raw eggs, as it presents a substantial risk of contamination by Salmonella spp.³ Furthermore, the absence of heat during salad cream production further exacerbates the susceptibility of consumers to Salmonella spp. infection. Manifesting in symptoms such as diarrhea, stomach ache, nausea, vomiting, and even mortality, the ramifications of Salmonella spp. infection are dire.^4,5,6 In light of escalating concerns regarding the safety of chemical preservatives employed to regulate microbial proliferation in food, there exists a burgeoning demand for natural alternatives to these agents. Consequently, the exploration of plant essential oils as a safer substitute for such additives has emerged as an area of intense focus.^7,8

The antimicrobial potential of essential oils derived from various spices and herbs, including finger root, clove, lemongrass, and cardamom, has been extensively demonstrated. However, the composition of essential oils in a species is influenced by numerous factors, including genotype-cultivar variations,⁹ the geographical region of cultivation,¹⁰ the plant parts selected for oil extraction,¹¹ and the specific extraction methods employed.¹² These essential oils exhibit remarkable efficacy against a wide range of pathogenic microorganisms.^13,14 According to Burt, ¹⁵ essential oils can be defined as aromatic oily liquids obtained from plant materials. Steam distillation is the common means to derive essential oil for commercial production. The mentioned finger root, clove, lemongrass and cardamom, examples among others, have been incorporated in human dishes for many centuries, and have been claimed for a long time to show a benefit for health improvement in the human body, some even being used in medical treatment for patients.¹⁶ Focusing on the antibacterial properties of those essential oils, finger root essential oil has been found to inhibit pathogens such as Listeria monocytogenes, Salmonella spp.,¹⁷ Citrobacter freundii, Escherichia coli, Klebsiella pneumonia and Serratia marcescens.¹⁸ Clove essential oil has been discovered to possess antimicrobial properties that can restrain the growth of Yersinia enterocolitica, E. coli, Pseudomonas aeruginosa, S. choleraesuis, Staphylococcus aureus, Bacillus cereus, L. monocytogenes and Enterococcus faecalis.¹⁹ Lemongrass essential oil can inhibit Bacillus cereus, B. subtilis, E. coli, S. aureus and K. pneumonia.²⁰ Moreover, cardamom essential oil inhibits B. subtilis, B. cereus, L. monocytogenes, S. aureus and S. typhimurium.²¹ Through hydrophobic interaction, essential oils can penetrate the bacterial cellular membranes and mitochondria, which can disrupt their structure, increase permeability, and eventually lead to cell leakage and destruction.¹⁵

Mathematical models play a crucial role in elucidating and predicting the growth and survival patterns of microorganisms within specific experimental conditions. However, it is important to acknowledge that the data derived from mathematical modelling merely represents predictions. Consequently, it is essential to validate these predicted outcomes by comparing them with empirical experimental data. The utilization of mathematical models to explicate and forecast microorganism growth and survival involves merging experimental data with mathematical frameworks.²² These models enable the anticipation of the future abundance or persistence of microorganisms, by employing mathematical equations that capture their respective growth or decline processes. Two fundamental types of equations are commonly employed for this purpose: linear equations and non-linear equations. The linear mathematical model, which encompasses the first-order kinetic model,^23,24 serves as a popular tool for explaining and predicting microbial growth and reduction. Both Weibull^25,26,27 and modified Gompertz^28,29,30,31 models are indeed widely employed mathematical models that are used to describe the temporal changes in microbial growth. These models consider various factors and parameters to accurately represent the growth patterns of microorganisms over time. The first-order kinetic model, being linear in nature, finds common application in forecasting microbial growth and reduction as it pertains to the studied temperature conditions. The Weibull model, on the other hand, is frequently employed to predict microbial survival and the decline phase, while non-linear models enable the explanation and prediction of microbial reduction in processes involving heat and those that do not involve heat.

Henceforth, the present study aimed to assess the efficacy of commercially available essential oils derived from four distinct spices, namely finger root, clove, lemongrass, and cardamom, both individually and in combination, as potential antibacterial agents against S. tphimurium. The evaluation involved determining the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the essential oils obtained from these spices. In addition, mathematical models, specifically the first-order kinetic, Weibull, and modified Gompertz models, were developed to forecast the survival of S. typhimurium following treatment with the essential oils, with consideration given to a storage temperature of 4 °C. These models were designed to enhance understanding and facilitate the management of the impact of essential oils on microbial growth and survival throughout the storage duration. Furthermore, this investigation explored the potential utilization of these essential oils in salad cream.

Materials and methods

Preparation of bacterial cultures for inhibitory tests

The Salmonella typhimurium strain (TISTR 1469) was preserved on tryptic soy agar (TSA) slants at a temperature of 5 °C. To prepare the active slants, a loopful of S. typhimurium was transferred into a tube containing 10 ml of tryptic soy broth (TSB), and subsequently incubated at 37 °C for a period of 18-24 hours. The turbidity of S. typhimurium suspensions was adjusted using sterile saline solution (0.85% NaCl) to match that of a McFarland Standard No. 0.5 (10⁸ CFU/ml), which had been modified following the methodology described by de Nova³² and the Clinical and Laboratory Standards Institute.³³

Preparation of essential oils from spices for inhibitory tests

Four essential oils of spices were used in this study: finger root (Boesenbergia pandurata (Roxb.) Schltr.) and lemongrass (Cymbopogon citratus (DC. ex Nees) Stapf) from AP Operation Co., Ltd. (Chonburi, Thailand), and clove (Syzygium aromaticum (L.) Merr. & Perry) and cardamom (Elettaria cardamum Maton) from Lapis Tropical Spa Products (Bangkok, Thailand). The finger root samples utilized in this study are sourced as fresh rhizomes, carefully hand-harvested directly from local farmers. Within the operations of AP Operation Co., Ltd., a specialized quality control department is tasked with the diligent evaluation of both the size and quality of these finger root rhizomes. Subsequently, an extensive cleaning process is undertaken to prepare them for the extraction of essential oil using a precise steam distillation method. The resulting essential oil is then meticulously packaged in 100 ml containers. Similarly, in the case of lemongrass, this company obtains fresh leaves from farmers through meticulous hand-harvesting. A dedicated unit within the company is responsible for the selection of lemongrass leaves based on criteria such as size, colour, and overall quality. These carefully chosen lemongrass leaves then undergo a thorough cleaning process in preparation for essential oil extraction, utilizing the same meticulous steam distillation method. The resulting lemongrass essential oil is also elegantly packaged, this time in 10 ml containers.

Lapis Tropical Spa Products, located in Bangkok, Thailand, operates as an intermediary for the procurement of essential oils, specifically clove and cardamom varieties. The clove essential oil samples are derived from freshly harvested flower buds, obtained from a specialized Indonesian enterprise engaged in essential oil extraction through the steam distillation method. The clove essential oil samples are carefully packaged in conveniently-sized 10 ml containers. In contrast, the cardamom essential oil samples are sourced from the desiccated seeds of cardamom plants cultivated and processed in India, also utilizing the steam distillation technique. These cardamom oil samples are presented in 10 ml containers.

The essential oils obtained from finger root, lemongrass, cloves, and cardamom possess a shelf life of two years. These oils can be suitably stored either refrigeration at 4 °C or storage at room temperature, maintained at 25 °C. All four essential oils were stored in amber glass bottles to protect them from UV light and reduce the potential degradation of the oils. The research study was conducted during the period from 2020 to 2022, during which all experiments were meticulously initiated and successfully concluded prior to the expiration of the two-year shelf life for each of the four types of essential oils. However, further research will focus on laboratory-based essential oil extraction to determine the optimal conditions, with a consideration of important factors such as extraction methods, temperature, solvent-to-plant ratio, and the type of solvent.

The preparation of essential oils from spices for inhibitory tests involved 15 treatments, as detailed in Table 1. In the case of combined essential oils, this study will collect essential oil samples to be blended together based on the combination ratios specified in Table 1. Subsequently, a volume of 50 μl from the blended essential oil was combined with 950 μl of 10% v/v DMSO, resulting in a final concentration of 50 μl/ml.

Table 1: Treatments, essential oils and their combination

Screening of essential oils using paper disk diffusion technique

Using Lorian’s method,³⁴ the entire surface of Mueller Hinton agar (MHA) was swabbed with a sterile cotton swab that had been dipped in an inoculum suspension of S. typhimurium. After that, 6 mm diameter sterile paper discs infused with essential oils from 15 different treatments were placed on the agar medium by slightly pressing them. The plates were incubated at 37 °C for 24 hours, and the diameters of the inhibition zones around the discs were measured using a digital caliper. To ensure accuracy, a negative control containing 10% v/v DMSO and a positive control containing 30 µg of tetracycline and 30 µg of chloramphenicol were added as well. The plates were left to rest at room temperature of 25 ^oC for 15 minutes before incubating to allow for excess pre-diffusion of essential oils.

Determination of minimum inhibitory concentration

The determination of the minimum inhibitory concentration (MIC) of each essential oil sample against S. typhimurium was conducted following the outlined procedure (modification of the Clinical and Laboratory Standards Institute).³³ Initially, 0.5 ml of Mueller Hinton broth (MHB) was prepared in tubes, which were subsequently lightly sealed and sterilized in an incubator. Once sterilized, the tubes were allowed to cool, after which the essential oil was added at a concentration of 50 µl/ml, with a volume of 0.5 ml. The essential oils derived from spices were then subjected to a two-fold serial dilution method, resulting in the following dilution sequence of essential oil concentrations: 25, 12.50, 6.250, 3.125, 1.5625, 0.781, 0.390, 0.195, 0.098, 0.049 and 0.025 μl/ml, respectively. Following the preparation of S. typhimurium, 0.5 ml of the bacterial culture was added to each respective test tube. A positive control tube was included, containing S. typhimurium without any essential oil, while negative controls were prepared without the presence of bacteria. All test tubes were then incubated at a temperature of 37 °C for a duration of 24 hours. Subsequently, the turbidity of the culture media was observed to determine the minimum concentration of essential oils required to inhibit bacterial growth, in comparison to the control test tubes. The MIC refers to the lowest concentration of essential oil necessary to entirely inhibit the growth of microorganisms. This concentration is determined through the observation of the absence of turbidity, indicating the absence of visible growth. The MIC value is expressed in microliters per milliliter (µl/ml) as a unit of essential oil concentration.

Determination of minimum bactericidal concentration at 37 °C and 4 °C

The determination of the minimal bactericidal concentration (MBC) was performed following a modified approach recommended by the Clinical and Laboratory Standards Institute.³³ The test tube samples showing no turbidity, as per the method described in the “Determination of minimum inhibitory concentration” section, were streaked onto TSA plates. The MBC of each essential oil was assessed at both 37 °C and 4 °C, representing the lowest concentration required to completely kill the microorganisms after a 24-hour incubation period. In this study, the MBC values are expressed in microliters per milliliter (µl/ml), serving as a unit for quantifying the concentration of essential oil.

Enumeration of surviving Salmonella typhimurium populations

Four single essential oils and combinations that showed the strongest inhibitory effect against S. typhimurium were selected and then prepared in MHB. MHB tubes containing essential oils at 1-, 4-, 16- and 64-fold the MBC were inoculated with a suspension of S. typhimurium including one positive control (S. typhimurium + MHB) to confirm that S. typhimurium could grow in MHB and one negative control (MHB) to ensure that the MHB was not contaminated. All samples in the tubes were incubated at 4 °C and stored under a controlled refrigerated temperature of 4 °C. The samples were analysed every 0.5 hr interval for 6 hr.

Mathematical models of survival curves

To describe the survival of S. typhimurium, mathematical models of S. typhimurium survival curves were obtained by creating graphs of the interaction of surviving S. typhimurium populations and time. This study selected three inactivation models, namely the first-order kinetic, Weibull, and modified Gompertz models.

First-order kinetic model

The first-order kinetic model is a linear model that can be used to predict microbial growth or reduction over time. It provides insights into the changes in the number of microorganisms at the experimental temperature.

where the variable N(t) represents the quantity of microorganisms present at a given time, t (log CFU/ml), the variable N₀ represents the initial quantity of microorganisms at the start of the experiment or at time zero (log CFU/ml), k is the survival rate of microorganisms (1/hr) and t is time (hr).

Weibull model

The Weibull model is a non-linear model used to predict survival or reduction of microorganisms.

where the variable N represents the quantity of microorganisms present at a given time, t (log CFU/ml), the variable, the variable N₀ represents the initial quantity of microorganisms at the start of the experiment or at time zero (log CFU/ml), b is the survival rate of microorganisms (1/hr), n is the shape of the survival curve and t is time (hr).³⁵

Modified Gompertz model

where the variable N represents the quantity of microorganisms present at a given time, t (log CFU/ml), the variable, the variable N₀ represents the initial quantity of microorganisms at the start of the experiment or at time zero (log CFU/ml), M is the number of microorganisms that enter the constant state (log CFU/ml), A is the survival rate of microorganisms (1/hr) and t is time (hr).²⁸

Quality of the model

The precision of the models was evaluated based on the adjusted coefficient of determination ( R²) and root mean square error (RMSE), which were calculated as follows.

where n is the number of trials.

Preparation of salad cream

Salad cream samples were prepared using a Philips VHR 2061 homogenizer in Indonesia. The ingredients used in the preparation included vinegar, sugar, sweetened condensed milk, ground pepper, salt, mustard, and eggs, constituting 14%, 19%, 8%, 1%, 1%, 1%, 47%, and 9% (w/w) of the salad cream, respectively. The ingredients were blended and homogenized for a duration of 2 minutes. Subsequently, the mixture was slowly incorporated into the vegetable oil phase while continuously stirring until a consistent and uniform texture was obtained.

Salmonella typhimurium analysis of salad cream samples

The analysis of S. typhimurium was modified based on the study conducted by Andrews.³⁶ Each sample of salad cream, weighing 25 g, was meticulously measured and placed in a sterile plastic stomacher bag containing 225 ml of sterile lactose broth. The mixture was then aseptically blended for 2 min. The pH of the mixture was assessed using test paper, and if necessary, adjusted to 6.8 ± 0.2 with sterile 1 N NaOH or 1 N HCl. The homogenized sample within the stomacher bag was incubated for 24 hours at 37 °C. After incubation, 1 ml of the mixture was transferred to 10 ml of Tetrathionate (TT) broth, vortexed, and then incubated at 37 °C for an additional 24 hours. A 3 mm loopful of the incubated TT broth was streaked onto xylose lysine desoxycholate (XLD) agar. Following incubation of the XLD agar at 37 °C for 24 hours, colonies suspected to be Salmonella were selected based on their characteristic appearance. Salmonella colonies typically exhibited pink or red coloration, with or without a black center on XLD agar. In cases where the colonies did not display these characteristics, colonies with a yellow colour and with or without a black center on XLD agar were selected. At least two colonies meeting these criteria were streaked onto tryptic soy agar (TSA) and incubated at 37 °C for 24 hours. Subsequently, biochemical tests were conducted to confirm the presence of Salmonella. These tests involved the utilization of sugars in triple sugar iron (TSI) agar, the production of the enzyme lysine decarboxylase in lysine iron agar (LIA), and the assessment of sulfur, indole, and motility in sulfide indole motility (SIM) medium. Following the biochemical tests, the cultures were further incubated at 37 °C for 24 hours before confirming the presence of Salmonella based on the biochemical results.

Microbiological analysis of salad cream samples

The microbiological analysis of salad cream samples was performed, utilizing a modified methodology based on the approach proposed by Maturin and Peeler.³⁷ After aseptically weighing 25 g of each salad cream sample into a sterile plastic stomacher, the sample was homogenized in 225 ml sterile peptone water (PW), beaten and blended for 1 min. To prepare decimal dilutions of 10⁻², 10⁻³, 10⁻⁴, and subsequent dilutions, 1 ml of the previous dilution was transferred to 9 ml dilution tubes using a sterile automatic pipette. Each dilution (1 ml) was pipetted into plate count agar (PCA). Using a sterile glass spreader, the diluted sample was evenly spread over the entire plate’s agar surface. The plates were then incubated for 24 hr at 37 °C.

Analysis of Salmonella typhimurium in salad cream incorporating essential oils from spices

Seven salad cream samples (650 g each) were prepared as shown in Table 2. The concentrations of essential oil which exhibited the best antimicrobial activity to kill S. typhimurium at 4 °C and during the storage period (at temperature of 4 °C) were used for further study. Subsequently, samples were subjected to microbiological analysis to determine the presence of S. typhimurium populations. The analysis was conducted at the onset of storage, followed by daily assessments up to day 7.

Table 2: Samples of salad cream prepared for testing the survival of Salmonella typhimurium

Analysis of microorganisms in salad cream incorporating essential oils from spices

Six salad cream samples (700 g each) which were prepared as shown in Table 3 – salad cream from a local market and supermarket, salad cream from Mae Hia market (shop 1), salad cream from Mae Hia market (shop 2) and salad cream from the Royal Project – were used to determine the number of microorganisms. Moreover, all salad cream samples were packed, stored at a temperature of 4 °C and analysed on days 0, 7 and 15 for the survival of S. typhimurium and every 3 days from 0 to 15 days for total microorganisms and pH value.

Table 3: Salad cream samples prepared for testing the survival of microorganisms

Sensory evaluation of salad cream incorporating essential oils from spices

A sensory laboratory was utilized to conduct the sensory analysis, wherein a trained sensory panel comprising of 30 assessors (18 women and 12 men aged between 20 and 32 years, all non-smokers) affiliated with the Faculty of Agro-Industry at Chiang Mai University. This panel assessed nine coded samples of salad cream, rating them on a 9-point hedonic scale based on attributes such as appearance, colour, aroma, and texture.

Expression of results and statistical analysis

Each experiment was conducted at least three times to ensure accuracy and reproducibility of results. The data obtained were subjected to statistical analysis using one-way ANOVA, and mean values were compared using Duncan’s mean comparison test. The statistical analyses were performed using SPSS 17.0 for Windows, and a significance level of 5% was set for the analyses.

Results and discussion

Test of ability of essential oils from spices to inhibit Salmonella typhimurium

The results for the ability of essential oils from spices to inhibit S. typhimurium at a concentration of 50 μl/ml using the paper disk diffusion technique on MHA indicated that essential oils from all four types of spices and their combinations exhibited good inhibitory ability against S. typhimurium. According to the findings presented in Table 4, the essential oil derived from clove exhibited the highest potency in inhibiting S. typhimurium compared to those from lemongrass, cardamom, and finger root, in descending order. The average diameter of the clear zone was 22.00 ± 0.50, 17.36 ± 0.69, 14.80 ± 0.45 and 10.80 ± 0.76 mm, respectively. As shown in Table 4, the combined clove and lemongrass essential oils showed the greatest inhibitory ability (19.39 mm). Furthermore, when essential oil from clove was combined with others with poor ability, a synergistic effect could be observed; for example, clove oil combined with finger root oil, clove oil combined with cardamom oil and clove oil combined with lemongrass oil.

Table 4: Antibacterial efficacy of spice-derived essential oils against Salmonella typhimurium via paper disk diffusion

Note: Tests used 6 mm diameter discs. Values are the average ± standard deviation. Statistically significant differences within the same column are denoted by different lowercase letters in superscript, with a significance level set at p < 0.05.

Antimicrobial activity of essential oils from spices against Salmonella typhimurium at 37 °C: Determining minimum inhibitory concentration and minimal bactericidal concentration

The results for the MIC and MBC of spice essential oils against S. typhimurium using a two-fold dilution technique are shown in Table 5. The best result was obtained for lemongrass essential oil, the second best for cardamom, clove and finger root oils. The MIC were 0.049, 0.390, 0.390 and 0.781 μl/ml and MBC were 0.049, 0.781, 0.781 and 0.781 μl/ml, respectively. For combined essential oils, lemongrass oil combined with cardamom oil showed the greatest activity, with an MIC of 0.195 μl/ml and MBC of 0.195 μl/ml.

The MBC is either greater than or equal to the MIC. This is because the MBC specifies the minimum concentration of the essential oil needed to completely eradicate the microorganism, while the MIC represents a preliminary value used to determine the essential oil concentration required to inhibit bacterial growth. The present result, shown in Table 5, showed that the MBC obtained is greater than or equal to the MIC, which is in agreement with the results of Snoussi.²¹ who studied the effect of cardamom essential oil on bacteria; their results revealed that the MBC value was greater than the MIC value. Also, Raybaudi-Massilia³⁸ studied the antimicrobial activity of essential oils (lemongrass, cinnamon, geraniol, palmarosa, clove, benzaldehyde) against S. enteritidis, E. coli and Listeria innocua using the agar broth dilution technique; they also revealed that the MBC was greater than the MIC.

Essential oils from spices can inhibit S. typhimurium because of the compounds they contain: clove essential oil has eugenol as its main constituent (70–80%)³⁹, finger root essential oil has 1,8-cineole and geraniol as its main constituents⁴⁰, lemongrass essential oil has α-citral and β-citral as its main constituents⁴¹ and cardamom essential oil had α-terpinyl acetate and 1,8-cineole as its main effective constituents.²¹ These compounds break the bacterial cell wall, leading to cell death.¹⁵

Table 5: Antimicrobial activity of essential oils from spices: minimum inhibitory concentration and maximum bactericidal concentration

Note: Values are the average ± standard deviation. Statistically significant differences within the same column are denoted by different lowercase letters in superscript, with a significance level set at p < 0.05.

Minimal bactericidal concentration of essential oils from spices against Salmonella typhimurium at 4 °C

As shown in Table 6, the essential oil for which the lowest concentration was required to kill S. typhimurium was that obtained from lemongrass, with an MBC of 0.781 µl/ml. The clove, finger root and lemongrass oils combined with cardamom oil had the same MBC of 3.125 µl/ml. The essential oil with the highest concentration required to kill S. typhimurium was that from cardamom, with the highest MBC of 6.250 µl/ml.

Table 6: Minimum bactericidal concentration of essential oils from spices at 4 °C

Note: Values are the average ± standard deviation. Statistically significant differences within the same column are denoted by different lowercase letters in superscript, with a significance level set at p < 0.05.

The MBC of essential oils from spices was found to be greater at 4°C compared to 37 °C, which is consistent with the experiments of Govaris⁴² who evaluated the inhibitory activity of oregano essential oil at concentrations of 0.6% v/v and 0.9% v/v against S. enteritidis in minced sheep meat during storage for 12 days at 4 °C and 10 °C. The results showed that oregano essential oil was more effective at inhibiting the bacteria in minced sheep meat at 10°C compared to 4 °C, with concentrations of 6% v/v and 0.9% v/v proving to be the most effective. In addition to investigating the inhibitory activity of essential oils, researchers have also explored the inhibitory effects of the active compounds found in these oils. Belda-Galbis⁴³ studied the ability of citral in essential oil from lemongrass to inhibit E. coli at 15, 30 and 37 °C and Listeria innocua at 8, 15, 30 and 37 °C. The results showed that the MIC of citral against E. coli at 30 and 37 °C was 0.325 µl/ ml, but at 15 °C it decreased to 3.000 µl/ml. As for the inhibitory activity against L. innocua, at 37 and 30 °C, the MIC was 0.300 µl/ml. At 15 °C, the MIC was 0.500 µl/ml and at 8 °C it was 1.000 µl/ml. The authors concluded that temperature has an effect on the ability of essential oils to inhibit and kill bacteria. Also, it affects the concentration of active substance in essential oils that are used to inhibit and kill bacteria because when the temperature drops, the microbial cells are stable and increased in strength, so that the essential oils or active substances in them penetrate the cell membrane in smaller quantities. Therefore, the concentration of essential oil and active substances in essential oils should be increased. In other words, the MIC and MBC of essential oil need to be increased as well.⁴⁴

Survival of Salmonella typhimurium at a storage temperature of 4 °C

Highly perishable food is usually stored at 4 °C. The antibacterial activity test in this experiment thus was of interest at this temperature. Five essential oils and their combination giving the highest activity (or lowest MBC) – finger root oil, clove oil, lemongrass oil, cardamom oil and lemongrass oil combined with cardamom oil – were selected for testing against S. typhimurium at 4 °C. The result showed that the concentration of finger root and clove essential oils required to kill S. typhimurium at 4 °C was four times the MBC at 37 °C (3.125 μl/ml) (Table 7). The corresponding concentrations for essential oils from cardamom, lemongrass and lemongrass combined with cardamom were 12.5, 0.781 and 3.125 μl/ml, respectively, representing a 16-fold increase from their MBC. Furthermore, complete antibacterial activity could be observed within 30 min for all tested essential oils except for lemongrass oil for which complete antibacterial activity was observed after 3 hr. The result could imply that temperature has an effect on the antibacterial activity of essential oils, namely, that the MBC at a lower temperature is greater than the MBC at a higher temperature to maintain the greatest efficacy for antibacterial activity. The temperature dependency of MBC was also reported by Govaris,⁴² who studied the inhibitory effect of 0.6% and 0.9% essential oil from oregano on S. enteritidis in ground lamb; their result indicated that keeping ground lamb at 10 °C inhibited germs better than at 4 °C.

The essential oils employed in this study exhibited inhibitory effects on S. typhimurium; however, further investigation is necessary to elucidate the specific constituents of essential oils responsible for this activity. Future research should aim to identify and evaluate the individual components of essential oils that effectively exert an effect on S. typhimurium.

Table 7: Inhibitory activity of essential oils from spices against Salmonella typhimurium at 4 °C

Mathematical models predicting the survival of Salmonella typhimurium using essential oils

Tables 8 and 9 present the parameters of various models employed to predict the survival of S. typhimurium in the presence of essential oils, as well as the coefficients of determination ( R²) and root mean square error (RMSE). The results indicated that the Weibull and modified Gompertz models were more suitable for describing the reduction in the number of surviving S. typhimurium compared to the first-order kinetic model. These findings are consistent with a previous study by Oliveira³⁵, which utilized the Weibull model to describe the number of S. enteritidis in raw mashed beef treated with essential oils from oregano and lemongrass during storage at 4 ± 2 °C for 6 days. Nikkhah⁴⁵ studied the ability of essential oils from basil, cinnamon, rosemary and marjoram to kill Penicillium expansum and Botrytis cinerea; their results showed that the modified Gompertz model could explain the reduction of number of P. expansum and B. cinerea.

Table 8: Modelling parameters for first-order kinetic, Weibull, and Modified Gompertz models.

The parameters presented in Table 8 offer quantitative insights into the behaviour of the models and aid in comprehending how the response variable evolves over time or under specific conditions. These parameter values are estimated through data fitting or statistical analysis to determine the optimal fit of the model to the observed data. In the context of the first-order kinetic model, the parameter “k” denotes the rate at which a reaction occurs. A higher “k” value signifies a faster reaction, while a lower value suggests a slower rate. For the Weibull model, the parameter “b” influences the shape of the curve and determines the rate of change of the response variable over time. A higher “b” value leads to a steeper increase or decrease in the response variable, resulting in a more rapid change. Conversely, a lower “b” value yields a more gradual change. Additionally, the “n” parameter in the Weibull model determines the time required for the response variable to reach a certain proportion. It affects the duration needed to achieve the specified level. A higher “n” value indicates a longer time for reaching the desired proportion, while a lower value suggests a shorter duration. The modified Gompertz model incorporates the “M” and “A” parameters. The maximum value of “M” represents the highest level that the response variable can reach. It characterizes the maximum growth or decay attainable by the response variable. On the other hand, the initial value of “A” denotes the starting point of the response variable before it begins to change or evolve. It signifies the initial level of the response at the onset of the process or reaction. A higher “A” value corresponds to a higher starting point, whereas a lower value indicates a lower initial level.

Therefore, mathematical models that accurately describe S. typhimurium survival offer numerous advantages across various domains. These models possess predictive power, enabling us to understand how S. typhimurium behaves under different conditions. Additionally, they facilitate the optimization of strategies for controlling S. typhimurium survival in healthcare and agriculture settings. By using mathematical models, we gain insights into the underlying mechanisms governing S. typhimurium survival. This knowledge can be utilized to enhance cost and time efficiency by reducing the necessity for extensive experimental work. Overall, mathematical models provide a quantitative framework that aids in understanding and managing S. typhimurium populations. They guide interventions, predict outcomes, and contribute to the development of effective measures for combating the threat of S. typhimurium.

However, in the further study, it is crucial to broaden the investigation by exploring a wider range of concentrations of spice essential oils. This comprehensive approach allows for a thorough examination of the factors influencing the survival rates of S. typhimurium. To gain a holistic understanding, it is recommended to incorporate mathematical modelling approaches in these investigations. This integration will provide valuable insights into the complex dynamics and interactions involved.

Table 9: Comparison of coefficients of determination and root mean square error of first-order kinetic, Weibull, and Modified Gompertz models

Anti-Salmonella typhimurium activity of essential oils from spices in salad cream

In the present study, during storage at 4 °C, no S. typhimurium was found in salad cream samples incorporating essential oils from spices (finger root, clove, lemongrass, cardamom and lemongrass combined with cardamom) and to which S. typhimurium had been added (as shown in Table 10). However, S. typhimurium was detected in salad cream samples with added S. typhimurium but without essential oils during a storage period of 7 days at 4 °C. Salmonella spp. are able to adapt to an inappropriate environment. For example, Salmonella spp. can grow or survive in salad cream samples during refrigerated storage 2–4 °C.³⁵ In addition, Salmonella spp. can survive for months at low temperatures (< 5 °C) and adapt to inappropriate external pH, responding to acid resistance by the process called acid tolerance response (ATR).⁴⁶ For the reasons above, the addition of essential oils from spices could inhibit the growth of S. typhimurium in salad cream samples during storage for 7 days at 4 °C.

Despite the significant impact on bacterial growth observed when incorporating specific components, namely ground pepper, and mustard, during the salad cream preparation process, these ingredients were deliberately included in the control sample of the salad cream. Consequently, the addition of essential oils to the salad cream in this study resulted in a considerable reduction in bacterial counts compared to the control sample, which lacked any essential oils.

Table 10: Salmonella typhimurium in salad cream with and without essential oils from spices during storage at 4 °C

x means S. typhimurium was not detected; / means S. typhimurium was detected.

Assessment of antimicrobial activity of spice-derived essential oils in salad cream

Salad cream samples containing essential oils from spices (finger root, clove, lemongrass, cardamom and lemongrass combined with cardamom), salad cream from a local market and supermarket, salad cream from Mae Hia market (shop 1), salad cream from Mae Hia market (shop 2) and salad cream from the Royal Project were used to determine the number of microorganisms and pH value. Additionally, the salad cream samples were packed, stored at 4 °C, and subjected to analysis on days 0, 7, and 15 to assess the survival of S. typhimurium. Total microorganisms and pH were analyzed at 3-day intervals between 0 and 15 days of storage. The results showed that S. typhimurium was not found in any of the salad cream samples containing essential oils from spices (finger root, clove, lemongrass, cardamom and lemongrass combined with cardamom) during 15 days of storage. The total number of microbials in salad cream from Mae Hia market (shop 1 and shop 2) and salad cream without essential oils from spices was higher than that in salad cream containing essential oils from spices and salad cream from the Royal Project Foundation at 12 and 15 days. However, all salad cream samples were in accordance with the Thai Community Products Standard for salad dressing products in Thailand (< 10,000 CFU/g). The approximate range of pH of all salad cream samples was 3.52–4.24. The salad cream from Mae Hia market (shop 1) had a higher range of pH (4.02–4.24) compared to other salad cream samples.

Sensory evaluation of salad cream incorporating essential oils from spices

Based on the consumer preference data presented in Table 11, there were no statistically significant differences (p < 0.05) observed between salad cream without essential oils from spices, salad cream with essential oils from spices (finger root, clove, lemongrass, cardamom and lemongrass combined with cardamom) and salad cream from the Royal Project Foundation. Furthermore, the colour and overall liking ratings for all salad cream samples did not demonstrate any significant differences (p < 0.05). In addition, the salad cream from the Royal Project Foundation had the lowest scores for appearance and texture. The salad cream without essential oils from spices had the highest odour scores.

Table 11: Mean scores for sensory acceptability of salad cream with and without essential oils from spices and salad cream from the Royal Project Foundation

Note: Values are the average ± standard deviation. Statistically significant differences within the same column are denoted by different lowercase letters in superscript, with a significance level set at p < 0.05.

Conclusions

The essential oils from finger root, clove, lemongrass, cardamom, and lemongrass combined with cardamom showed strong inhibitory activity against S. typhimurium. At 4 °C, the required concentrations of finger root and clove essential oils to kill S. typhimurium were four times higher than the MBC at 37 °C (3.125 μl/ml). Cardamom, lemongrass, and lemongrass combined with cardamom essential oils exhibited corresponding concentrations of 12.5, 0.781, and 3.125 μl/ml, representing a 16-fold increase from their MBC. Complete inactivation occurred within 30 minutes for most essential oils, except for lemongrass oil, which took 3.5 hours. The modified Gompertz model fit the surviving number of S. typhimurium when using finger root essential oil, while the Weibull and modified Gompertz models best described the decrease in surviving bacteria when inhibited by clove, lemongrass, cardamom, and lemongrass combined with cardamom essential oils.

During 15-day storage at 4 °C, salad cream containing essential oils from spices (finger root, clove, lemongrass, cardamom, and lemongrass combined with cardamom) and S. typhimurium exhibited no detectable presence of the bacteria, and the overall microbe count decreased. These findings suggest the potential of spice essential oils as natural preservatives. Sensory evaluations comparing appearance, colour, odour, texture, and overall liking between salad creams without essential oils, with essential oils, and from the Royal Project Foundation showed no significant differences (p < 0.05). Hence, salad cream containing essential oils from spices can be used for commercial production.

The obtained results demonstrate the significant inhibitory and antiseptic effects of essential oils derived from spices against S. typhimurium. However, further investigations are necessary to delve into the chemical composition of finger roots, cloves, lemongrass, and cardamom essential oils. This research should prioritize the identification and characterization of specific bioactive constituents responsible for the inhibitory and eradication properties against S. typhimurium. Additionally, it is essential to conduct studies utilizing a broader range of concentrations for spice essential oils in order to comprehensively assess their impact on the survival rates of S. typhimurium. By employing mathematical modelling approaches, a more thorough understanding of the underlying factors influencing these survival rates can be achieved.

Acknowledgements

The authors acknowledge the Faculty of Agro-Industry, Chiang Mai University for financial support.

Conflicts of Interest

The author(s) declares no conflict of interest.

Funding Sources

The author(s) received no financial support for the research, authorship, and/or publication of this article.

References

1. Teshome E., Forsido S. F., Rupasinghe H. P. V., Keyata E. O. Potentials of natural preservatives to enhance food safety and shelf life: A review. Hindawi Sci World J. 2022. https://doi.org/10.1155/2022/9901018.
   CrossRef
2. Yin M., Chen M., Yanagisawa T., Matsuoka R., Xi Y., Tao N., Wang X. Physical properties, chemical composition, and nutritional evaluation of common salad dressings. Front Nutr. 2022; 9: 978648. https://doi.org/10.3389/fnut.2022.978648.
   CrossRef
3. Keerthirathne T. P., Ross K., Fallowfield H., Whiley H. A review of temperature, pH, and other factors that influence the survival of Salmonella in mayonnaise and other raw egg products. Pathogens. 2016; 5: 63. https://doi.org/10.3390/pathogens5040063.
   CrossRef
4. Chen, H.M.; Wang, Y.; Su, L.H.; Chiu, C.H. Nontyphoid salmonella infection: Microbiology, clinical features, and antimicrobial therapy. Pediatr Neonatol. 2013; 54: 147–152. https://doi.org/10.1016/j.pedneo.2013.01.010.
   CrossRef
5. Zha L., Shari Garrett S., Sun J. Salmonella infection in chronic inflammation and gastrointestinal cancer. Diseases. 2019; 7: 28. https://doi.org/10.3390/diseases7010028
   CrossRef
6. Popa G. L., Popa M. I. Salmonella spp. infection – a continuous threat worldwide. Germs. 2020; 11(1): 88-96. https://doi.org/10.18683/germs.2021.1244.
   CrossRef
7. Saeed K., Pasha I., Chughtai, M. F. J., Ali Z., Bukhari H., Zuhair M. Application of essential oils in food industry: Challenges and innovation. J Essent Oil Res. 2022; 34: 97-110. https://doi.org/10.1080/10412905.2022.2029776.
   CrossRef
8. Hanková K., Lupomˇeská P., Nový P., Všeteˇcka D., Klouˇcek P., Kouˇrimská L., Hlebová M., Božik M. Effect of conventional preservatives and essential oils on the survival and growth of Escherichia coli in vegetable sauces: A comparative study. Foods. 2023; 12: 2832. https://doi.org/10.3390/foods12152832.
   CrossRef
9. Maria G. A., Riccardo N. Citrus bergamia, Risso: the peel, the juice and the seed oil of the bergamot fruit of Reggio Calabria (South Italy). Emir J Food Agric. 2020; 522-532. https://doi.org/10.9755/ejfa.2020.v32.i7.2128.
   CrossRef
10. Sharma N., Singh B., Wani M. S., Gupta R. C., Habeeb T. H. Determination of the volatile composition in essential oil of Azadirachta indica A. Juss from different areas of north indian plains by gas chromatography/mass spectrometry (GC/MS). Anal Chem Lett. 2021; 11:1: 73-82. https://doi.org/10.1080/22297928.2021.1877195.
    CrossRef
11. Gurkana H., Hayaloglub A. A. Changes in volatiles and essential oil composition of three organs (leaf, stem and flower) of purple basil (Ocimum basilicum L.) by GC–MS combined with multivariate statistical approach. Food Chem Adv. 2023; 2: 100292. https://doi.org/10.1016/j.focha.2023.100292.
    CrossRef
12. Chatterjee S., Gupta S., Variyar P.S. Comparison of essential oils obtained from different extraction techniques as an aid in identifying aroma significant compounds of Nutmeg (Myristica fragrans). Nat Prod Commun. 2015; 10(8): 1443-1446. https://doi.org/10.1177/1934578X1501000833
    CrossRef
13. Swain S. S., Paidesetty S. K., Padhy R. N., Hussain T. Nano-technology platforms to increase the antibacterial drug suitability of essential oils: A drug prospective assessment. OpenNano. 2003; 9: 100115. https://doi.org/10.1016/j.onano.2022.100115.
    CrossRef
14. Cordery A., Rao A. P., Ravishankar S. Antimicrobial activities of essential oils, plant extracts and their applications in foods- A review. J Agric Environ Sci. 2018; 7(2): 76-89. https://doi.org/10.15640/jns.v7n2a9
    CrossRef
15. Burt S. Essential oils: Their antibacterial properties and potential applications in foods-a review. Food Microbiol. 2004; 94(3): 223-253. https://doi.org/10.1016/j.ijfoodmicro.2004.03.022.
    CrossRef
16. Kaefer C. M., John A. Milner J. A. The role of herbs and spices in cancer prevention. J Nutr Biochem. 2008; 19(6): 347-361. https://doi.org/10.1016/j.jnutbio.2007.11.003.
    CrossRef
17. Thongson C., Davidson P. M., Mahakarnchanakul W., Vibulsrethm P. Antimicrobial effect of Thai spices against Listeria monocytogenes and Salmonella typhimurium DT104. J Food Prot. 2005; 68(10): 2054-2058. https://doi.org/10.4315/0362-028x-68.10.2054.
    CrossRef
18. Nanasombat S., Lohasupthawee P. Antibacterial activity of crude ethanolic extracts and essential oils of spices against Salmonella and other enterobacteria. KMITL Sci J. 2005; 5: 527–538.
19. Goni P., Lopez P., Sanchez C., Gomez-Lus R., Becerril R., Nerin C. Antimicrobial activity in the vapour phase of a combination of cinnamon and clove essential oils. Food Chem. 2009; 116(4): 982-989. https://doi.org/10.1016/j.foodchem.2009.03.058.
    CrossRef
20. Mohd I. N., Bashir A. F., Ebenezar J., Javid A. B. Antibacterial activity of lemongrass (Cymbopogon citratus) oil against some selected pathogenic bacterias. Asian Pac J Trop Med. 2010; 3(7): 535-538. https://doi.org/10.1016/S1995-7645(10)60129-0.
    CrossRef
21. Snoussi M., Noumi E., Dehmani A., Flamini G., Aouni M., Alsieni M., Al-sieni A. Chemical composition and antimicrobial activities of Elettaria cardamomum L. (Manton) essential oil: A high activity against a wide range of food borne and medically important bacteria and fungi. JCBPS. 2016; 6(1): 248-259.
22. Lebert I., Robles-Olvera V., Lebert A. Application of polynomial models to predict growth of mixed cultures of Pseudomonas spp. and Listeria in meat. Int J Food Microbiol. 2000; 61: 27-39. https://doi.org/10.1016/S0168-1605(00)00359-7
    CrossRef
23. Mcclure P. J., Beaumont A. L., Sutherland J. P., Roberts T. A. Predictive modeling of growth of Listeria monocytogenes the effects on growth of NaCl, pH, storage temperature and NaNO2. Int J Food Microbiol. 1997; 34: 221-232. https://doi.org/10.1016/s0168-1605(96)01193-2.
    CrossRef
24. Cirelli G. L., Consoli S., Juanico M. Modelling Escherichia coli concentration in a wastewater reservoir using an operational parameter MRT%FE and first order kinetics. J Environ Manage. 2009; 90: 604-614. https://doi.org/10.1016/j.jenvman.2007.12.015
    CrossRef
25. Albert I., Mafart P. A modified Weibull model for bacterial inactivation. Int J Food Microbiol. 2005; 100: 197-211. https://doi.org/10.1016/j.ijfoodmicro.2004.10.016
    CrossRef
26. Gonzalez M., Skandamis P. N., Hanninen M. A modified Weibull model for describing the survival of Campylobacter jejuni in minced chicken meat. Int J Food Microbiol. 2009; 136: 52-58. https://doi.org/10.1016/j.ijfoodmicro.2009.09.022.
    CrossRef
27. Evrendilek G. A., Balasubramaniam, V. M. Inactivation of Listeria monocytogenes and Listeria innocua in yogurt drink applying combination of high-pressure processing and mint essential oils. Food Control. 2011; 22: 1435-1441. https://doi.org/10.1016/j.foodcont.2011.03.005
    CrossRef
28. Vandepitte V., Quataert, P., Rore D. H., Verstraete W. Evaluation of the gompertz function to model survival of bacteria introduced into soils. Soil Biol Biochem. 1995; 27(3): 365-372. https://doi.org/10.1016/0038-0717(94)00158-W
    CrossRef
29. Erkmen O. Mathematical modeling of Salmonella typhimurium inactivation under high hydrostatic pressure at different temperatures. Food Bioprod Process. 2009; 87: 68-73. https://doi.org/10.1016/j.fbp.2008.05.002
    CrossRef
30. Nikkhah M., Hashemi, M., Najafi, M. B. H., Farhoosh, R. Synergistic effects of some essential oils against fungal spoilage on pear fruit. Int J Food Microbiol. 2017; 257: 285-294. https://doi.org/10.1016/j.ijfoodmicro.2017.06.021
    CrossRef
31. Menezes N. M. C., Martins, W. F., Longhi, D. A., Aragao, G. M. F. D. Modeling the effect of oregano essential oil on shelf-life extension of vacuum-packed cooked sliced ham. Meat Sci. 2018; 139: 113-119. https://doi.org/10.1016/j.meatsci.2018.01.017
    CrossRef
32. de Nova, P. J. G., Carvajal, A., Prieto M., Rubio P. In vitro susceptibility and evaluation of techniques for understanding the mode of action of a promising non-antibiotic citrus fruit extract against several pathogens. Front. Microbiol. 2009; 10: 884. https://doi.org/ 10.3389/fmicb.2019.00884
    CrossRef
33. Clinical and Laboratory Standard Institute (CLSI). Methods for dilution antimicrobial susceptibility test for bacteria that grow aerobically; Approved Standard-Eight edition. CLSI documents M07-A8. Wayne, PA. Clinical and Laboratory Standard Institute; 2009.
34. Lorian E. V. Antibiotic in laboratory medicine. 3rd edition. Philadelphia, USA: Lippincott Williams & Wilkins; 1991.
35. Oliveira T. L. C. D., Soares R. D. A., Piccoli R. H. A Weibull model to describe antimicrobial kinetics of oregano and lemongrass essential oils against Salmonella enteritidis in ground beef during refrigerated storage. Meat Sci. 2013; 93(3): 645-651. https://doi.org/10.1016/j.meatsci.2012.11.004.
    CrossRef
36. Andrews W. H., Wang H., Jacobson A., Hammack T. Chapter 5: Salmonella. In: Bacteriological Analytical Manual (BAM). Washington DC, USA: Food and Drug Administration (FDA); 2018. https://www.fda.gov/food/laboratory-methods-food/bam-chapter-5-Salmonella. (22 February 2018).
37. Maturin L., Peeler J. T. Chapter 3: Aerobic plate count. In: Bacteriological Analytical Manual (BAM). 8th edition. Silver Spring, Berlin: Food and Drug Administration (FDA); 2018. https://www.fda.gov/food/laboratory-methods-food/bam-chapter-3-aerobic-plate-count. (22 February 2018).
38. Raybaudi-Massilia R. M., Mosqueda-Melgar J., Martín-Belloso O. Antimicrobial activity of essential oils on Salmonella enteritidis, Escherichia coli, and Listeria innocua in fruit juices. J Food Prot. 2006; 69(7): 1579-1586. https://doi.org/10.4315/0362-028X-69.7.1579.
    CrossRef
39. Shelef L. A. Antimicrobial effects of spices. J Food Saf. 1983; 6: 29-44. https://doi.org/10.1111/j.1745-4565.1984.tb00477.x.
    CrossRef
40. Phanthong P., Lomarat P., Chomnawang M. T., Bunyapraphatsara N. Antibacterial activity of essential oils and their active components from Thai spices against food borne pathogens. ScienceAsia. 2013; 39(5): 472-476. https://doi.org/10.2306/scienceasia1513-1874.2013.39.472.
    CrossRef
41. Onawunmi G. O. Effect of dimethyl sulfoxide on the anti-bacterial activity of lemongrass oil. Microbios Lett. 1987; 36: 105-111.
42. Govaris A., Solomakos N., Pexara A., Chatzopoulou P. S. The antimicrobial effect of oregano essential oil, nisin and their combination against Salmonella enteritidis in minced sheep meat during refrigerated storage. Int J Food Microbiol. 2010; 137(2-3): 175-180. https://doi.org/10.1016/j.ijfoodmicro.2009.12.017.
    CrossRef
43. Belda-Galbis C. M., Leufven A., Martinez A., Rodrigo D. Quantitative assessment of citral antimicrobial potential at different temperatures. In: Méndez-Vilas A. Microbial pathogens and strategies for combating them: science, technology and education. Badajoz, Spain: Formatex Research Center; 2013: 1257-1264.
44. D’Amico S., Collins T., Marx J. C., Feller G., Gerday C. Psychrophilic microorganisms: Challenges for life. EMBO Reports. 2006; 7(4): 385-389. https://doi.org/10.1038/sj.embor.7400662.
    CrossRef
45. Nikkhah M., Hashemi M., Najafi M. B. H., Farhoosh R. Synergistic effects of some essential oils against fungal spoilage on pear fruit. Int J Food Microbiol. 2017; 257: 285-294. https://doi.org/10.1016/j.ijfoodmicro.2017.06.021.
    CrossRef
46. Greenacre E. J., Brocklehurst T. F., Waspe C. R., Wilson D. R., Wilson P. D. G. Salmonella enterica serovar typhimurium and Listeria monocytogenes acid tolerance response induced by organic acids at 20 °C: optimization and modeling. Appl Environ Microbiol. 2003; 69(7): 3945-3951. https://doi.org/10.1128/AEM.69.7.3945-3951.2003
    CrossRef